Methods of inhibition using glycosyl sulfotransferase-3

ABSTRACT

A novel human glycosylsulfotransferase expressed in high endothelial cells (GST-3) and polypeptides related thereto, as well as nucleic acid compositions encoding the same, are provided. The subject polypeptides and nucleic acid find use in a variety of applications, including research, diagnostic, and therapeutic agent screening applications. Also provided are methods of inhibiting selectin mediated binding events and methods of treating disease conditions associated therewith, particularly by administering an inhibitor of at least one of GST-3 or KSGal6ST, or homologues thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 09/190,911filed Nov. 12, 1998, now U.S. Pat. No. 6,365,365, which is acontinuation-in-part of application Ser. No. 09/045,284 filed on Mar.20, 1998, now U.S. Pat. No. 6,265,192, the disclosure of which is hereinincorporated by reference.

INTRODUCTION

1. Field of the Invention

The field of the invention is cell adhesion, particularly selectinmediated cell adhesion, as well as the treatment of disease conditionsrelated thereto.

2. Background of the Invention

Sulfotransferases are enzymes that catalyze the transfer of a sulfatefrom a donor compound to an acceptor compound, usually placing thesulfate moiety at a specific location on the acceptor compound. Thereare a variety of different sufotransferases which vary in activity, i.e.with respect to the donor and/or acceptor compounds with which theywork. Known sulfotransferases include those acting on carbohydrate:

heparinileparan sulfate N-sulfotransferase (NST), chondroitin 6/keratan6 sulfate sulfotransferase (C6ST/KSST); galactosylceramide3′-sulfotransferase; heparan sulfate 2-sulfotranserase (Iduronic acid);HNK-1 sulfotransferase (3-glucuronic acid); heparan sulfateD-glucosamino 3-O-sulfotransferase (3-OST); etc., as well as thoseacting on phenols, steroids and xenobiotics: aryl sulfotransferase I &II, hydroxy-steroid sulfotransferases I, II & III,dehydroepiandrosterone (DHEA); etc. Sulfotransferases play a centralrole in a variety of different biochemical mechanisms, as the presenceof a sulfate moiety on a particular ligand is often required for aparticular activity, e.g. binding.

The presence of a sulfate moiety on selectin ligands has been shown tobe important for selectin binding to occur. See Imai et al., Nature(1993) 361:555-557 and Imai et al., Glycoconjugate J. (1993) 10:34-39,as well as U.S. Pat. No. 5,695,752. Several selectin ligands have, todate, been identified. The L-selectin endothelial ligands in mouse thathave been identified are: CD34, GlyCAM-1, MAdCAM-1 and sgp200. Inaddition, PSGL-1 has been identified as a leukocyte ligand for P-, E-,and L-selectin. Endothelial ligands for L-selectin in humans are stillpoorly defined, but include CD34 and podocalyxin.

Selectin mediated binding plays an important and prominent role in avariety of biological processes. Selectins are lectin like cell adhesionmolecules that mediate leukocyte-endothelial, leukocyte-leukocyte,leukocyte-platelet, platelet-endothelial and platelet-plateletinteractions. One critical biological process in which selectin mediatedbinding plays a role is the maintenance of immune surveillance.

Maintenance of immune surveillance depends on the constant recirculationof lymphocytes from the blood through the vascular wall into the tissuesand eventually back into the blood. Lymphocyte recruitment from theblood into all secondary lymphoid organs (except the spleen) as well asinto many sites of chronic inflammation is mediated by a specializedpostcapillary venule called a high endothelial venule. These vessels aredefined by the distinct, cuboidal morphology of their endothelial cellsand their lulminal presentation of ligands for the leukocyte adhesionmolecule, L-selectin. This lectin-like adhesion molecule is expressed onall classes of leukocytes in the blood and is responsible for theinitial tethering and rolling of a leukocyte on the endothelium prior tosubsequent integrin mediated firm arrest and transrugration.

Although selectin mediated binding events play a critical role in normalphysiological processes, disease conditions do exist for which it isdesired to regulate or modulate, e.g. limit or prevent, the amount ofselectin mediated binding that occurs. Such conditions include: acute orchronic inflammation; autoimmnune and related disorders, tissuerejection during transplantation, and the like.

As the above conditions all result from selectin mediated bindingevents, there is great interest in the elucidation of the mechanismsunderlying such binding events. There is also great interest in theidentification of treatment methodologies for these and related diseaseconditions, as well, the identification of active agents for usetherein.

As such, there is continued interest in the identification ofparticipants in the selectin binding mechanism, including enzymaticagents, and the elucidation of their role(s) in selectin mediatedbinding events, as well as the development of therapies for diseaseconditions arising from such binding events.

Relevant Literature

Chondroitin-6-sulfotransferase is disclosed in EP 821 066, as well as inFukuta et al., “Molecular Cloning and Characterization of Human KeratanSulfate Gal-6-Sulfotransferase,” J. Biol. Chem. (Dec. 19, 1997) 272:32321-32328; Habuchi et al., “Enzymatic Sulfation of Galactose Residueof Keratan Sulfate by Chondroitin 6-Sulfotransferase,” Glycobiology(January 1996) 6:51-57; Habuchi et al., “Eqnzyatic Sulfation ofGalactose Residue of Keratan Sulfate by Chondroitin 6-Sulfate byChondroitin 6-Sulfotransferase,” Glycobiology (January 1996) 6:51-57;Fukuta et al., “Molecular Cloning and Expression of Chick ChondrocyteChondroitin 6-Sulfotransferase,” J. Biol. Chem. (1995) 270: 18575-18580;and Habuchi et al., “Purification of Chondroitin 6-SulfotransferaseSecreted from Cultured Chick Embryo Chondrocytes,” J. Biol. Chem. (1993)268: 21968-21974.

References providing background information on selectin mediated bindinginclude: Baumhueter et al., “Binding of L-Selectin to the VascularSialomucin CD34,” Science (Oct. 15, 1993): 436-438; Boukerche et al., “AMonoclonal Antibody Directed Against a Granule Membrane Glycoprotein(GMP-140/PADGEM, P-selectin, CD62P Inhibits Ristocetin-Induced PlateletAggregation,” Br. J. Haematology (1996) 92: 442-451; Celi et al.,“Platelet-Leukocyte-Endothelial Cell Interaction on the Blood VesselWall,” Seminars in Hematology (1997) 34: 327-335; Frenette et al.,“Platelets Roll on Stimulated Endotheliumn In Vivo: An InteractionMediated by Endothelial P-selectin,” Proc. Natl. Acad. Sci. USA (August1995) 52:7450-7454; Girard & Springer, “High Endothelial Venules (HEVs):Specialized Endothelium for Lymphocyte Migration,” Immun. Today (1995)16: 449-457; Hemmerich et al., “Sulfation Dependent Recognition of HighEndothelial Venules (HEV)Ligands by L-Selectin and Meca79, andAdhesion-Blocking Monoclonal Antibody,” J. Exp. Medicine (December 1994)180: 2219-2226; 262 Lasky et al., “An Endothelial Ligand for L-Selectinis a Novel Mucin-Like Molecule,” Cell (Jun. 12, 1992) 69:927-938; Rosen& Bertozzi, “The Selectins and Their Ligands,” Current Opinion in CellBiology (1994) 6: 663-673; and Sawada et al., “Specific Expression of aComplex Sialyl Lewis X Antigen On High Endothelial Venules of HumanLymph Nodes: Possible Candidate for L-selectin Ligand,” Biochem.Biophys. Res. Comm. (May 28, 1993) 193: 337-347; as well as U.S. Pat.No. 5,580,862.

U.S. Pat. No. 5,695,752 describes methods of treating inflammationthrough administration of sulfation inhibitors.

SUMMARY OF THE INVENTION

A novel human glycosyl sulfosferase (GST-3 or HEC-GlcNAc6ST) andpolypeptides related thereto, as well as nucleic acid compositionsencoding the same, are provided. The subject polypeptide and nucleicacid compositions find use in a variety of applications, includingresearch, diagnostic, and therapeutic agent screening applications, aswell as in treatment therapies. Also provided are methods of inhibitingselectin mediated binding events and methods of treating diseaseconditions associated therewith, particularly by administeringinhibitors of GST-3 and/or KSGal6ST (or an HEC specific homologuethereof).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the cDNA sequence and amino acid sequence of HumanGST-3. The cDNA sequence is SEQ ID NO:02 and the amino acid sequence isSEQ ID NO:01.

FIG. 2 provides the hydropathy plot for GST-3 (HEC-GlcNAc6ST). Thehydrophobicity was calculated by the method of Kyte and Doolittle J.Mol. Biol. (1982) 157, 105-32; using a window of 11 amino acids.

FIG. 3 provides a representation of the derivation of primers that wereemployed in homology PCR on HEV-cDNA. Peptide sequences GST-1 and GST-2were aligned with several known sulfotransferases retrieved from theGenbank database using the Clustal W (Thompson et al., Nuc. Acids Res.(1994) 22: 4673-4680) algoritm Three consensus regions A, B, & C wereidentified. These are depicted above in order 5′ to 3′ (N-terminal toC-terminal) with the intermittent and flanking regions of less or nohomology truncated. Highly degenerate forward primers A+ and B+ andreverse primers B− and C− containing toggle bases as well as inosinewere designed as described in the experimental section. These primersencode a maxi number of permutations at the amino-acid level in order toaccommodate most patterns highlighted above in red or brown

FIG. 4 provides a representation of the protein domain structure ofC6ST/KSST and GST-1, GST-2 and GST-3.

FIG. 5 provides the alignment of regions of high conservation amonghuman carbohydrate 6-sulfotransferases (Gal-6, GlcNAc6 and GalNAc-6).Protein sequences were aligned using the Clustal W algorithm (Thompsonet al., 1994, supra). Black shading indicates identity at that residueamong at least three of the sequences, grey shading indicatessimilarity.

FIG. 6 provides a graphical representation of the amount of sulfate(SO₄) incorporation into GlyCAM-1 in COS7 cells cotransfected with: (a)GlyCAM-1/Ig expression plasmid plus GST-3 expression plasmid; and (b)GlyCAM-1/Ig expression plasmid plus vector lacking GST-3 insert; and (c)untransfected cells.

FIG. 7 provides the sulfated O-linked carbohydrate chains of GlyCAM-1.Oligosaccharides bearing the 6-sulfo sialyl Lewis x (sLe^(x)) and the6′-sulfo sLe^(x) motif, which extend from the core 2 structure areshown. The presence of the 6′,6-disulfo sLe^(x) motif is stronglysuspected. Structures of the more complex O-linked chains of GlyCAM-1remain to be determined.

FIG. 8 provides a graphical representation of the results of an assaydesigned to study the sulfation of synthetic acceptors by HEC-GlcNAc6ST.Microsomal extracts from COS cells transfected with HEC-GlcNAc6ST cDNAor vector cDNA (mock) were reacted with [³⁵S]-PAPS and the disaccharideor trisaccharide acceptors (see Table 3). After a 2 hour incubation at37° C., radiolabeled acceptor was isolated by reversed-phasechromatography and incorporation was quantified by liquid scintillationcounting. Each datapoint represents the average of triplicatedeterminations. The error bars indicate SEMs.

FIG. 9 provides the results obtained when the disaccharide acceptorsulfated by extracts of HEC-GlcNAc6ST transfectants (see A) wassubjected to mild acid hydrolysis. The hydrolysate was analysed by highpH anion exchange chromatography using the following standards: 1,GlcNAc-3-SO₃ ⁻; 2, [³⁵S]-SO₄ ²⁻; 3, Gal-4-SO₃ ⁻; 4, Gal-3-SO₃ ⁻; 5,GlcNAc-6-SO₃ ⁻; 6; Gal-6-SO₃ ⁻. The hydrolysate from the disaccharideacceptor sulfated by HEC-GlcNAc6ST transfectant microsomes (--Δ--)showed only two radioactive peaks, that comigrated with free sulfate(left peak) and GlcNAc6-sulfate (right peak). Hydrolysate fromdisaccharide acceptor sulfated by mock transfectant microsomes (--●--)showed only free sulfate.

FIG. 10 provides the results of an assay designed to study the sulfationof GlyCAM-1/IgG conferred by HEC-GlcNAc6ST and KSGal6ST cDNAs. COS cellswere transfected with combinations of plasmids encoding GlyCAM-1/IgG andthe two sulfotansferases as indicated. Transfected cells were culturedin the presence of [³⁵S]-sulfate and recombinant GlyCAM-1/IgG wasisolated from the conditioned medium by passage over protein A-agarose.1% of the captured material was subjected to SDS-PAGE and the remainderwas processed for analysis of sulfated carbohydrate. Densitometricquantification of the Coomassie Blue-stained bands following SDS-PAGEshowed that each lane, except the control lane without GlyCAM-1/IgGplasmid, contained approximately equal amounts of GlyCAM-1/IgG. Analysisof sulfated carbohydrate in GlyCAM-1/IgG coexpressed with HEC-GlcNAc6STor KSGal6ST is provided in the graph of FIG. 10. Sulfated mono- anddisaccharides derived from [³⁵S]-sulfate labeled GlyCAM-1/IgG producedby COS cells transfected with HEC-GlcNAc6ST (--Δ--) or KSGal6ST (--●--),as described above, were analyzed by high pH anion exchangechromatography after acid hydrolysis. The standards were as follows: 1,GlcNAc-3SO₃ ⁻; 2, [³⁵S]—SO₄ ²⁻; 3, Galβ1-4[SO₃ ⁻; 6]GlcNAc; 4, [SO₃⁻-6]Galβ1-4GlcNAc; 5, Gal-4SO₃ ⁻; 6, Gal-3SO₃ ⁻; 7, GlcNAc6SO₃ ⁻; 8,Gal-6SO₃ ⁻.

FIGS. 11A to 11C provides the results of an assay in whichCHO/FTVII/C2GnT cells were transfected with different combinations ofcDNAs encoding human CD34, HEC-GlcNAc6ST and KSGal6ST. cDNAs encodingFTVII and C2GnT were included in all transfection mixtures to ensuresufficient expression levels of these enzymes. Cells were stained withthe L-selectin/IgM chimera to detect ligand activity. FIGS. 11A and 11Bare histograms showing L-selectin/IgM staining for the followingtransfections: CD34 cDNA only ( , A and B); CD34/HEC-GlcNAc6ST/KSGal6STcDNAs ( , A and B); CD34/HEC-GlcNAc6ST/KSGal6ST cDNAs with staining donein the presence of Mel-14 mAb ( , A); HEC-GlcNAc6/KSGal6ST cDNAs but noCD34 cDNA ( , B). The secondary staining reagent when used alone showedstaining equal to that observed when Mel-14 mAb was used as aninhibitor. The isotype-matched control for Mel-14 showed no inhibitoryeffect. FIG. 11C provides a the results of a two color analysis showingCD34 expression (y-axis, staining with CD34-PE mAb) and L-selectinligand activity (x-axis, FITC) in cells trafsfected with CD34 cDNA andcDNAs encoding HEC-GlcNAc6ST and KSGal6ST, alone or in combination asindicated. The horizontal bar is set such that all cells staining withthe isotype matched control for the CD34 mAb are included in the lowerquadrants. The vertical bar is set to indicate the L-selectin/IgMstaining of cells in which no sulfotransferase cDNA was included in thetransfection mixture (lower left panel). The fraction of positive cells(as a percentage of the total) in each quadrant is indicated. The meanfluorescence intensity (MFI) for the cells in the upper right quadrantsis indicated. The CD34 mAb did not interfere with staining by theL-selectin/IgM chimera.

FIGS. 12 to 14 provide the results from rolling and tetheringexperiments which were conducted to elucidate the role of GST-3 andKSGal6ST in the biosynthesis of functional L-selectin ligands, asdescribed in greater detail in the experimental section infra.

DETAILED DESCRIPTION OF THE INVENTION

A novel human glycosyl transferase expressed in high endothelial cells(HEC) (i.e. GST-3 or HEC-GlcNAc6ST) and polypeptides related thereto, aswell as nucleic acid compositions encoding the same, are provided. Thesubject polypeptide and/or nucleic acid compositions find use in avariety of different applications, including research, diagnostic, andtherapeutic agent screening/discovery/ preparation applications. Alsoprovided are methods of inhibiting selectin mediated binding events andmethods of treating disease conditions associated therewith,particularly by administering an inhibitor of GST-3 and/or KSGal6ST, oran HEC specific homologue thereof.

Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Polypeptide Compositions

A novel human glycosylsulfotransferase expressed in high endothelialcells (HEC), as well as polypeptide compositions related thereto, areprovided. The term polypeptide composition as used herein refers to boththe full length human protein as well as portions or fragments thereof.Also included in this term are variations of the naturally occurringhuman protein, where such variations are homologous or substantiallysimilar to the naturally occurring protein, as described in greaterdetail below, as well as corresponding homologs from non-human species,such as other mammalian species. In the following description of thesubject invention, the terms GST-3 and HEC-GlcNAc6ST are used to refernot only to the human form of this novel sulfotransferase, but also tohomologs thereof expressed in non-human species.

The novel human glycosylsulfotransferase enzyme of the subject inventionhas been named human glycosyl sulfotransferase 3 or huGST-3 orHEC-GlcNAc6ST. huGST-3 is a type 2 membrane protein having a relativelyshort transmembrane domain and a short amino-terminal cytoplasmic tail.huGST-3 has a 31% amino acid sequence identity with CS6T/KSST (Habuchiet al., J. Biol. Chew (1995) 240:4172-4179) as measured by using the“GAP” program (part of the Wisconsin Sequence Analysis Package availablethrough the Genetics Computer Group, Inc. (Madison Wis.)), where theparameters are: Gap weight 12; length weight:4. huGST-3 is capable ofsulfating selectin ligands, particularly L-selectin ligands, e.g.GlyCAM-1. By sulfating selectin ligands is meant that huGST-3 is capableof catalyzing the transfer of a sulfate group from a donor compound to aposition on a selectin ligand precursor as acceptor compound. Donorcompounds from which huGST-3 obtains sulfate groups for transfer toacceptor ligand compounds include 3′-phosphoadenosine 5′-phosphosulfate(PAPS) and the like. Selectin ligands capable of being sulfated throughhuGST-3 action include E-, P- and L-selectin ligands, particularlyL-selectin ligands, such as GlyCAM-1, CD34, MAdCAM-1, Sgp200,podocalyxin, and the like. huGST-3 is strongly predicted to haveGlcNAc6-0-sulfotransferase (N-actylglucosamine-6-O-sulfotransferase)activity.

Human GST-3 is a 386 amino acid protein having an amino acid sequence asshown in FIG. 1 and identified as SEQ ID NO:01. huGST-3 has a molecularweight based on its amino acid of about 45 kDa to 46 kDa, and morespecifically from about 45100 to 45200 dalton, and specifically 45104dalton (using DNA Strider 1.2 software). Since GST-3 is glycosylated,its true molecular weight is greater, and is likely to be in the rangefrom about 45 to 85 kDa, and more likely from about 50 kDa to 65 kDa.Expression of GST-3 in humans is highly restricted. For example, huGST-3is expressed in HEC but not tonsillar lymphocytes, or primary culturedhuman umbilical vein endothelial cells (HUVEC).

In addition to the huGST-3, also provided are GST-3 proteins that havethe same expression pattern in humans as huGST-3, i.e. are highlyrestricted and expressed in HEC but not HUVEC or lymphocytes. huGST-3homologs or proteins (or fragments thereof) from nonhuman species arealso provided, including mammals, such as: rodents, e.g. mice, rats;domestic animals, e.g. horse, cow, dog, cat; and humans, as well asnon-mammalian species, e.g. avian, and the like. By homolog is meant aprotein having at least about 35%, usually at least about 40% and moreusually at least about 60% amino acid sequence identity to the huGST-3protein.

Also provided are GST-3 proteins that are substantially identical to thehuGST-3 protein, where by substantially identical is meant that theprotein has an amino acid sequence identity to the sequence of huGST-3of at least about 35%, usually at least about 40% and more usually atleast about 60%.

Also provided are KSGal6ST homologues that are selectively expressed inHEC. The nucleotide and amino acid sequence for KSGal6ST is reported inFukuta et al., J. Biol. Chem. (Dec. 19, 1997) 272:32321-32328. Thesubject HEC specific KSGal6ST homologues have a sequence that issubstantially identical to KSGal6ST, where by substantially identical ismeant that the protein has an amino acid sequence identity to thesequence of KSGal6ST of at least about 35%, usually at least about 40%and more usually at least about 60%.

The proteins of the subject invention (e.g. huGST-3 or a homologthereof; an HEC specific KSGal6ST homologue) are present in anon-naturally occurring environment, e.g. are separated from theirnaturally occurring environment. In certain embodiments, the subjectproteins are present in a composition that is enriched for subjectprotein as compared to its naturally occurring environment. For example,purified GST-3 is provided, where by purified is meant that GST-3 ispresent in a composition that is substantially free of non-GST-3proteins, where by substantially free is meant that less than 90%,usually less than 60% and more usually less than 50% of the compositionis made up of non-GST-3 proteins. The proteins of the subject inventionmay also be present as an isolate, by which is meant that the protein issubstantially free of other proteins and other naturally occurringbiologic molecules, such as oligosaccharides, polynucleotides andfragments thereof, and the like, where substantially free in thisinstance means that less than 70%, usually less than 60% and moreusually less than 50% of the composition containing the isolated proteinis some other naturally occurring biological molecule. In certainembodiments, the proteins are present in substantially pure form, whereby substantially pure form is meant at least 95%, usually at least 97%and more usually at least 99% pure.

In addition to the naturally occurring proteins, polypeptides which varyfrom the naturally occurring proteins are also provided, e.g. GST-3polypeptides. By GST-3 polypeptide is meant an amino acid sequenceencoded by an open reading frame (ORF) of the GST-3 gene, described ingreater detail below, including the full length GST-3 protein andfragments thereof, particularly biologically active fragments and/orfragments corresponding to functional domains, e.g. acceptor bindingsite (postulated to be the most 5′ consensus region A (see experimentalsection infra), the donor binding site, e.g. VRYEDL, and the like; andincluding fusions of the subject polypeptides to other proteins or partsthereof. Fragments of interest will typically be at least about 10 aa inlength, usually at least about 50 aa in length, and may be as long as300 aa in length or longer, but will usually not exceed about 1000 aa inlength, where the fragment will have a stretch of amino acids that isidentical to the subject protein of at least about 10 aa, and usually atleast about 15 aa, and in many embodiments at least about 50 aa inlength.

The subject proteins and polypeptides may be obtained from naturallyoccurring sources or synthetically produced. Where obtained fromnaturally occurring sources, the source chosen will generally depend onthe species from which the protein is to be derived. For example,huGST-3 is generally derived from endothelial cells of high endothelialvenules (HEV) of human secondary lymphoid organs, such as tonsils. Thesubject proteins may also be derived from synthetic means, e.g. byexpressing a recombinant gene encoding protein of interest in a suitablehost, as described in greater detail below. Any convenient proteinpurification procedures may be employed, where suitable proteinpurification methodologies are described in Guide to ProteinPurification, (Deuthser ed.) (Academic Press, 1990). For example, alysate may prepared from the original source, e.g. HEC or the expressionhost, and purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, and the like.

Nucleic Acid Compositions

Also provided are nucleic acid compositions encoding GST-3 proteins orfragments thereof, as well as the HEC-specific KSGal6ST homologues ofthe present invention. By GST-3 nucleic acid composition is meant acomposition comprising a sequence of DNA having an open reading framethat encodes GST-3, i.e. a GST-3 gene, and is capable, under appropriateconditions, of being expressed as GST-3. Also encompassed in this termare nucleic acids that are homologous or substantially similar oridentical to the nucleic acids encoding GST-3 proteins. Thus, thesubject invention provides genes encoding huGST-3 and homologs thereof.The human GST-3 gene has the nucleic acid sequence shown in FIG. 1 andidentified as SEQ ID NO:02, infra.

The source of homologous genes may be any species, e.g., primatespecies, particularly human; rodents, such as rats and mice, canines,felines, bovines, ovines, equines, yeast, nematodes, etc. Betweenmammalian species, e.g., human and mouse, homologs have substantialsequence similarity, e.g. at least 75% sequence identity, usually atleast 90%, more usually at least 95% between nucleotide sequences.Sequence similarity is calculated based on a reference sequence, whichmay be a subset of a larger sequence, such as a conserved motif, codingregion, flanking region, etc. A reference sequence will usually be atleast about 18 nt long, more usually at least about 30 nt long, and mayextend to the complete sequence that is being compared. Algorithms forsequence analysis are known in the art, such as BLAST, described inAltschul et al. (1990) J. Mol. Biol. 215:403-10 (using defaultsettings). The sequences provided herein are essential for recognizingGST-3-related and homologous proteins in database searches.

Nucleic acids encoding the GST-3 protein and GST-3 polypeptides of thesubject invention may be cDNA or genomic DNA or a fragment thereof. Theterm “GST-3 gene” shall be intended to mean the open reading frameencoding specific GST-3 proteins and polypeptides, and GST-3 introns, aswell as adjacent 5′ and 3′ non-coding nucleotide sequences involved inthe regulation of expression, up to about 20 kb beyond the codingregion, but possibly further in either direction. The gene may beintroduced into an appropriate vector for extrachromosomal maintenanceor for integration into a host genome.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons and 3′ and 5′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns, when present, being removed by nuclear RNAsplicing, to create a continuous open reading frame encoding a GST-3protein.

A genomic sequence of interest comprises the nucleic acid present,between the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include the 3′ and 5′untranslated regions found in the mature MRNA. It may further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ or 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller, and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ or 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue and stage specific expression.

The nucleic acid compositions of the subject invention may encode all orapart of the subject GST-3 protein Double or single stranded fragmentsmay be obtained from the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be of at least 15 nt, usually at least 18 nt or 25 nt,and may be at least about 50 nt.

The GST-3 genes arc isolated and obtained in substantial purity,generally as other than an intact chromosome. Usually, the DNA will beobtained substantially free of other nucleic acid sequences that do notinclude a GST-3 sequence or fragment thereof, generally being at leastabout 50%, usually at least about 90% pure and are typically“recombinant”, i.e. flanked by one or more nucleotides with which it isnot normally associated on a naturally occurring chromosome.

Preparation of GST-3 Polypeptides

In addition to the plurality of uses described in greater detail infollowing sections, the subject nucleic acid compositions find use inthe preparation of all or a portion of the GST-3 polypeptides, asdescribed above. For expression, an expression cassette may be employed.The expression vector will provide a transcriptional and translationalinitiation region, which may be inducible or constitutive, where thecoding region is operably linked under the trancriptional control of thetranscriptional initiation region, and a transcriptional andtranslational termination region. These control regions may be native toa GST-3 gene, or may be derived from exogenous sources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor the production of fusion proteins, where the exogenous fissionpeptide provides additional functionality, i.e. increased proteinsynthesis, stability, reactivity with defined antisera, an enzymemarker, e.g. -galactosidase, etc.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, and up to the completeopen reading flame of the gene. After introduction of the DNA, the cellscontaining the construct may be selected by means of a selectablemarker, the cells expanded and then used for expression.

GST-3 proteins and polypeptides may be expressed in prokaryotes oreukaryotes in accordance with conventional ways, depending upon thepurpose for expression. For large scale production of the protein, aunicellular organism, such as E. coli, B. subtilis, S. cerevtsiae,insect cells in combination with baculovirus vectors, or cells of ahigher organism such as vertebrates, particularly mammals, e.g. COS 7cells, may be used as the expression host cells. In some situations, itis desirable to express the GST-3 gene in eukaryotic cells, where theGST-3 protein will benefit from native folding and post-translationalmodifications. Small peptides can also be synthesized in the laboratory.Polypeptides that are subsets of the complete GST-3 sequence may be usedto identify and investigate parts of the protein important for function.

Uses of the Subject GST-3 Polypeptide and Nucleic Acid Compositions

The subject polypeptide and nucleic acid compositions find use in avariety of different applications, including research, diagnostic, andtherapeutic agent screening/discovery/preparation applications, as wellas therapeutic compositions.

Research Applications

The subject nucleic acid compositions find use in a variety of researchapplications. Research applications of interest include: theidentification of huGST-3 homologs; as a source of novel promoterelements; the identification of GST-3 expression regulatory factors; asprobes and primers in hybridization applications, e.g. PCR; theidentification of expression patterns in biological specimens; thepreparation of cell or animal models for GST-3 function; the preparationof in vitro models for GST-3 function; etc.

Homologs of GST-3 are identified by any of a number of methods. Afragment of the provided cDNA may be used as a hybridization probeagainst a cDNA library from the target organism of interest, where lowstringency conditions are used. The probe may be a large fragment, orone or more short degenerate primers. Nucleic acids having sequencesimilarity are detected by hybridization under low stringencyconditions, for example, at 50° C. and 6×SSC (0.9 M sodium chloride/0.09M sodium citrate) and remain bound when subjected to washing at 55° C.in 1×SSC (0.15 M sodium chloride/0.015 M sodium citrate). Sequenceidentity may be determined by hybridization under stringent conditions,for example, at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/01.5mM sodium citrate). Nucleic acids having a region of substantialidentity to the provided GST-3 sequences, e.g. allelic variants,genetically altered versions of the gene, etc., bind to the providedGST-3 sequences under stringent hybridization conditions. By usingprobes, particularly labeled probes of DNA sequences, one can isolatehomologous or related genes.

The sequence of the 5′ flanking region may be utilized for promoterelements, including enhancer binding sites, that provide fordevelopmental regulation in tissues where GST-3 is expressed. The tissuespecific expression is useful for determining the pattern of expression,and for providing promoters that mimic the native pattern of expression.Naturally occurring polymorphisms in the promoter region are usefull fordetermining natural variations in expression, particularly those thatmay be associated with disease.

Alternatively, mutations may be introduced into the promoter region todetermine the effect of altering expression in experimentally definedsystems. Methods for the identification of specific DNA motifs involvedin the binding of transcriptional factors are unknown in the art, e.g.sequence similarity to known binding motifs, gel retardation studies,etc. For examples, see Blackwell et al (1995), Mol. Med. 1:194-205;Mortlock et al. (1996), Genome Res. 6:327-33; and Joulin and Richard-Foy(1995), Eur. J. Biochem. 232:620-626.

The regulatory sequences may be used to identify cis acting sequencesrequired for transcriptional or translational regulation of GST-3expression, especially in different tissues or stages of development,and to identify cis acting sequences and trans-acting factors thatregulate or mediate GST-3 expression. Such transcription ortranslational control regions may be operably linked to a GST-3 gene inorder to promote expression of wild type or altered GST-3 or otherproteins of interest in cultured cells, or in embryonic, fetal or adulttissues, and for gene therapy.

Small DNA fragments are useful as primers for PCR, hybridizationscreening probes, etc. Larger DNA fragments, i.e. greater than 100 ntare useful for production of the encoded polypeptide, as described inthe previous section. For use in amplification reactions, such as PCR, apair of primers will be used. The exact composition of the primersequences is not critical to the invention, but for most applicationsthe primers will hybridize to the subject sequence under stringentconditions, as known in the art. It is preferable to choose a pair ofprimers that will generate an amplification product of at least about 50nt, preferably at least about 100 nt. Algorithms for the selection ofprimer sequences are generally known, and are available in commercialsoftware packages. Amplification primers hybridize to complementarystrands of DNA, and will prime towards each other.

The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature. Briefly, DNA or mRNA is isolatedfrom a cell sample. The MRNA may be amplified by RT-PCR, using reversetranscriptase to form a complementary DNA strand, followed by polymerasechain reaction amplification using primers specific for the subject DNAsequences. Alternatively, the mRNA sample is separated by gelelectrophoresis, transferred to a suitable support, e.g. nitrocellulose,nylon, etc., and then probed with a fragment of the subject DNA as aprobe. Other techniques, such as oligonucleotide ligation assays, insitu hybridizations, and hybridization to DNA probes arrayed on a solidchip may also find use. Detection of mRNA hybridizing to the subjectsequence is indicative of GST-3 gene expression in the sample.

The sequence of a GST-3 gene, including flanking promoter regions andcoding regions, may be mutated in various ways known in the art togenerate targeted changes in promoter strength, sequence of the encodedprotein, etc. The DNA sequence or protein product of such a mutationwill usually be substantially similar to the sequences provided herein,i.e. will differ by at least one nucleotide or amino acid, respectively,and may differ by at least two but not more than about ten nucleotidesor amino acids. The sequence changes may be substitutions, insertions,deletions, or a combination thereof. Deletions may further includelarger changes, such as deletions of a domain or exon. Othermodifications of interest include epitope tagging, e.g. with the FLAGsystem, HA, etc. For studies of subcellular localization, fusionproteins with green fluorescent proteins (GFP) may be used.

Techniques for in vitro mutagenesis of cloned genes are known Examplesof protocols for site specific mutagenesis may be found in Gustin et al.(1993), Biotechniques 14:22; Barany (1985), Gene 37:111-23; Colicelli etal. (1985), Mol. Gen. Genet. 199:537-9; and Prentli et al. (1984), Gene29:303-13. Methods for site specific mutagenesis can be found inSambrook et at, Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp. 15.3-15.108; Weiner eta. (1 993), Gene 126:35-41; Sayers et al.(1992), Biotechniques 13:592-6; Jones and Wuinistorfer (1992),Biotechniques 12:528-30; Barton et al. (1990), Nucleic Acids Res18:7349-55; Marotti and Tomich (1989), Gene Anal. Tech. 6:67-70; and Zhu(1989), Anal Biochem 177:120-4. Such mutated genes may be used to studystructure-function relationships of GST-3, or to alter properties of theprotein that affect its function or regulation.

The subject nucleic acids can be used to generate transgenic, non-humananimals or site specific gene modifications in cell lines. Transgenicanimals may be made through homologous recombination, where the normalgst-3 locus is altered. Alternatively, a nucleic acid construct israndomly integrated into the genome. Vectors for stable integrationinclude plasmids, retroviruses and other animal viruses, YACs, and thelike.

The modified cells or animals are useful in the study of gst-3 functionand regulation. For example, a series of small deletions and/orsubstitutions may be made in the host's native gst-3 gene to determinethe role of different exons in oncogenesis, signal transduction, etc. Ofinterest are the use of gst-3 to construct transgenic animal models forcancer, where expression of GST-3 is specifically reduced or absentSpecific constructs of interest include anti-sense gst-3, which willblock GST-3 expression, expression of dominant negative gst-3 mutations,and over-expression of GST-3 genes. Where a gst-3 sequence isintroduced, the introduced sequence may be either a complete or partialsequence of a gst-3 gene native to the host, or may be a complete orpartial gst-3 sequence that is exogenous to the host animal, e.g., ahuman GST-3 sequence. A detectable marker, such as lac Z may beintroduced into the gst-3 locus, where upregulation of gst-3 expressionwill result in an easily detected change in phenotype.

One may also provide for expression of the gst-3 gene or variantsthereof in cells or tissues where it is not normally expressed, atlevels not normally present in such cells or tissues, or at abnormaltimes of development.

DNA constructs for homologous recombination will comprise at least aportion of the human GST-3 gene or of a gst-3 gene native to the speciesof the host animal wherein the gene has the desired geneticmodification(s), and includes regions of homology to the target locus.DNA constructs for random integration need not include regions ofhomology to mediate recombination. Conveniently, markers for positiveand negative selection are included. Methods for generating cells havingtargeted gene modifications through homologous recombination are knownin the art. For various techniques for transfecting mammalian cells, seeKeown et al. (1990), Meth. Enzymol. 185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allogeneicor congenic grafts or transplants, or in in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc., e.g. to determine the effectof a candidate drug on GST-3 activity.

The availability of a number of components in the leukocyte traffickingmechanism, such as GlyCAM-1, L-selectin and the subject GST-3 enzyme,and the like, allows in vitro reconstruction of the mechanism, i.e. theproduction of an in vitro model.

Diagnostic Applications

Also provided are methods of diagnosing disease states based on observedlevels of GST-3 or the expression level of the GST-3 gene in abiological sample of interest. Samples, as used herein, includebiological fluids such as blood, cerebrospinal fluid, tears, saliva,lymph, dialysis fluid, semen and the like; organ or tissue culturederived fluids; and fluids extracted from physiological tissues. Alsoincluded in the term are derivatives and fractions of such fluids. Thecells may be dissociated, in the case of solid tissues, or tissuesections may be analyzed. Alternatively a lysate of the cells may beprepared.

A number of methods are available for determining the expression levelof a gene or protein in a particular sample. Diagnosis may be performedby a number of methods to determine the absence or presence or alteredamounts of normal or abnormal GST-3 in a patient sample. For example,detection may utilize staining of cells or histological sections withlabeled antibodies, performed in accordance with conventional methods.Cells are permeabilized to stain cytoplasmic molecules. The antibodiesof interest are added to the cell sample, and incubated for a period oftime sufficient to allow binding to the epitope, usually at least about10 minutes. The antibody may be labeled with radioisotopes, enzymes,fluorescers, chemiluminescers, or other labels for direct detection.Alternatively, a second stage antibody or reagent is used to amplify thesignal. Such reagents are well known in the art. For example, theprimary antibody may be conjugated to biotin, with horseradishperoxidase-conjugated avidin added as a second stage reagentAlternatively, the secondary antibody conjugated to a flourescentcompound, e.g. fluorescein, rhodamine, Texas red, etc. Final detectionuses a substrate that undergoes a color change in the presence of theperoxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

Alternatively, one may focus on the expression of GST-3. Biochemicalstudies may be performed to determine whether a sequence polymorphism ina GST-3 coding region or control regions is associated with disease.Disease associated polymorphisms may include deletion or truncation ofthe gene, mutations that alter expression level, that affect theactivity of the protein, etc.

Changes in the promoter or enhancer sequence that may affect expressionlevels of GST-3 can be compared to expression levels of the normalallele by various methods known in the at Methods for determiningpromoter or enhancer strength include quantitation of the expressednatural protein; insertion of the variant control element into a vectorwith a reporter gene such as β-galactosidase, luciferase,chloramphenicol acetyltaansferase, etc. that provides for convenientquantitation; and the like.

A number of methods are available for analyzing nucleic acids for thepresence of a specific sequence, e.g. a disease associated polymorphismWhere large amounts of DNA are available, genomic DNA is used directly.Alternatively, the region of interest is cloned into a suitable vectorand grown in sufficient quantity for analysis. Cells that express GST-3may be used as a source of mRNA, which may be assayed directly orreverse transcribed into cDNA for analysis. The nucleic acid may beamplified by conventional techniques, such as the polymerase chainreaction (PCR), to provide sufficient amounts for analysis. The use ofthe polymerase chain reaction is described in Saiki, et al. (1985),Science 239:487, and a review of techniques may be found in Sambrook, etal. Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp.14.2-14.33. Alternatively, various methods are known in the art thatutilize oligonucleotide ligation as a means of detecting polymorphisms,for examples see Riley et al. (1990), Nucl. Acids Res. 18:2887-2890; andDelahunty et al. (1996), Am. J. Hum. Genet. 58:1239-1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerydirin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein(HEXA), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, ahaptens, etc. having ahigh affity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid, e.g. amplified or cloned fragment, is analyzedby one of a number of methods known in the art The nucleic acid may besequenced by dideoxy or other methods, and the sequence of basescompared to a wild-type GST-3 sequence. Hybridization with the variantsequence may also be used to determine its presence, by Southern blots,dot blots, etc. The hybridization pattern of a control and variantsequence to an array of oligonucleotide probes immobilized on a solidsupport, as described in U.S. Pat. No. 5,445,934, or in WO 95/35505, mayalso be used as a means of detecting the presence of variant sequences.Single strand conformational polymorphism (SSCP) analysis, denaturinggradient gel electrophoresis (DGGE), and heteroduplex analysis in gelmatrices are used to detect conformational changes created by DNAsequence variation as alterations in electrophoretic mobility.Alternatively, where a polymorphism creates or destroys a recognitionsite for a restriction endonuclease, the sample is digested with thatendonuclease, and the products size fractionated to determine whetherthe fragment was digested. Fractionation is performed by gel orcapillary electrophoresis, particularly acrylamide or agarose gels.

Screening for mutations in GST-3 may be based on the functional orantigenic characteristics of the protein. Protein truncation assays areuseful in detecting deletions that may affect the biological activity ofthe protein. Various immunoassays designed to detect polymorphisms inGST-3 proteins may be used in screening. Where many diverse geneticmutations lead to a particular disease phenotype, functional proteinassays have proven to be effective screening tools. The activity of theencoded GST-3 protein may be determined by comparison with the wild-typeprotein.

Diagnostic methods of the subject invention in which the level of GST-3expression is of interest will typically involve comparison of the GST-3nucleic acid abundance of a sample of interest with that of a controlvalue to determine any relative differences, where the difference may bemeasured qualitatively and/or quantitatively, which differences are thenrelated to the presence or absence of an abnormal GST-3 expressionpattern A variety of different methods for determine the nucleic acidabundance in a sample are know to those of skill in the art, whereparticular methods of interest include those described in: Pietu et al.,Genome Res. (June 1996) 6: 492-503; Zhao et al., Gene (Apr. 24, 1995)156: 207-213; Soares , Curr. Opin. Biotechnol. (October 1997) 8:542-546; Raval, J. Pharmacol Toxicol Methods (November 1994) 32:125-127; Chalifour et al., Anal. Biochem (Feb. 1, 1994) 216: 299-304;Stolz & Tuan, Mol. Biotechnol. (December 19960 6: 225-230; Hong et al.,Bioscience Reports (1982) 2: 907; and McGraw, Anal. Biochem. (1984) 143:298. Also of interest are the methods disclosed in WO 97/27317, thedisclosure of which is herein incorporated by reference.

Screening Assays

The subject GST-3 polypeptides (as well as the HEC-specific KSGal6SThomologues) find use in various screening assays designed to identifytherapeutic agents. Thus, one can use a cell model such as a host cell,e.g. COS7 cell, which has been cotransfected with a selectin ligandcDNA, e.g. GlyCAM-1 or CD34 and a GST-3 vector. One can then label thetransfectants with a labeled sulfate, e.g. ³⁵ S-labeled sulfate, andcompare the amount of sulfate incorporation into GlyCAM-1 or CD34 in thepresence and absence of a candidate inhibitor compound. Alternatively,in a cell-free enzyme activity assay, recombinant GST-3 polypeptide maybe combined with ³⁵S-labeled sulfate donor such as [³⁵ S]-PAPS,candidate inhibitor compound, and an acceptor molecule, which may be asynthetic carbohydrate mimicking structures found in mature and/orimmature L-selectin ligands, or a simple nucleophile capable ofaccepting sulfate (such as phenolic compunds, and the like). The amountof [³⁵S]-sulfate transferred to the receptor by the candidate agent isthen determined by counting the acceptor-associated radioactivity orproduct quantitation with an antibody specific for the sulfatedacceptor, or in a suitable scintillation proximity assay format.Alternatively, the candidate inhibitor compound may also be combinedwith a selectin, a non-sulfated selectin ligand precursor, GST-3 and asulfate donor compound under physiological conditions and the resultantamount of ligand which is capable of binding to the selectin isdetermined. Depending on the particular method, one or more of, usuallyone of; the specified components may be labeled, where by labeled ismeant that the components comprise a detectable moiety, e.g. afluorescent or radioactive tag, or a member of a signal producingsystem, e.g. biotin for binding to an enzyme-streptavidin conjugate inwhich the enzyme is capable of converting a substrate to a chromogenicproduct.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used.

The above screening methods may be designed a number of different ways,where a variety of assay configurations and protocols may be employed,as are known in the art. For example, one of the components may be boundto a solid support, and the remaining components contacted with thesupport bound component. The above components of the method may becombined at substantially the same time or at different times.Incubations are performed at any suitable temperature, typically between4 and 40° C. Incubation periods are selected for optimum activity, butmay also be optimized to facilitate rapid high-throughput screening.Typically between 0.1 and 1 hours will be sufficient. Following thecontact and incubation steps, the subject methods will generally, thoughnot necessarily, further include a washing step to remove unboundcomponents, where such a washing step is generally employed whenrequired to remove label that would give rise to a background signalduring detection, such as radioactive or fluorescently labelednon-specifically bound components. Following the optional washing step,the presence of bound selectin-ligand complexes will then be detected.

A variety of different candidate agents may be screened by the abovemethods. Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

GST-3 Nucleic Acid and Polypeptide Therapeutic Compositions

The nucleic acid compositions of the subject invention also find use astherapeutic Ad agents in situations where one wishes to enhance GST-3activity in a host. The GST-3 genes, gene fragments, or the encodedGST-3 protein or protein fragments are useful in gene therapy to treatdisorders associated with GST-3 defects. Expression vectors may be usedto introduce the GST-3 gene into a cell. Such vectors generally haveconvenient restriction sites located near the promoter sequence toprovide for the insertion of nucleic acid sequences. Transcriptioncassettes may be prepared comprising a transcription initiation region,the target gene or fragment thereof, and a transcriptional terminationregion. The transcription cassettes may be introduced into a variety ofvectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus and thelike, where the vectors are able to transiently or stably be maintainedin the cells, usually for a period of at least about one day, moreusually for a period of at least about several days to several weeks.

The gene or GST-3 protein may be introduced into tissues or host cellsby any number of routes, including viral infection, microinjection, orfusion of vesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al. (1992), Anal Biochem205:365-368. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”as described in the literature (see, for example, Tang et al. (1992),Nature 356:152-154), where gold microprojectiles are coated with theDNA, then bombarded into skin cells.

Methods of Modulating Selectin Mediated Binding Events

Also provided are methods of regulating, including modulating andinhibiting, selectin mediated binding events. The selectin receptor ofthe selectin mediated binding event will generally be a receptor whichbinds to a sulfated ligand under physiological conditions and is amember of the selectin family of receptors that have an amino terminalC-type lectin domain followed by an EFG-like domain, a variable numberof short consensus repeats known as SCR, CRP or sushi domains, and sharegreater than 50% homology in their lectin and EFG domains. Of interestis the modulation of selectin binding events in which the selectin isL-, P-, or E-selectin. Of particular interest are L-selecting mediatedbinding events.

Where the selectin mediated binding event occurs in vivo in a host, inone embodiment an effective amount of active agent that modulates theactivity, usually reduces the activity, of GST-3 in vivo, isadministered to the hosl In another embodiment, the modulating agent istargeted to KSGal6ST, or an HEC-specific homologue thereof. In yetanother embodiment, one or more agents are administered that effectivelymodulate both the KSGal6ST or related activity and the GST-3 activity.The active agent may be a variety of different compounds, including anaturally occurring or synthetic small molecule compound, an antibody,fragment or derivative thereof, an antisense composition, and the like.

Naturally occurring or synthetic small molecule compounds of interestinclude numerous chemical classes, though typically they are organicmolecules, preferably small organic compounds having a molecular weightof more than 50 and less than about 2,500 daltons. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

Also of interest as active agent are antibodies that at least reduce, ifnot inhibit, the target activity in the host (e.g. the GST-3 activityand/or the KSGal6ST activity). Suitable antibodies are obtained byimmunizing a host animal with peptides comprising all or a portion ofthe target protein. Suitable host animals include mouse, rat sheep,goat, hamster, rabbit, etc. The origin of the protein immunogen may bemouse, human, rat, monkey etc. The host animal will generally be adifferent species than the immunogen, e.g. human GST-3 used to immunizemice, etc.

The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part ofGST-3, where these residues contain the post-translation modifications,such as glycosylation, found on the native target protein. Immunogenscomprising the extracellular domain are produced in a variety of waysknown in the art, e.g. expression of cloned genes using conventionalrecombinant methods, isolation from HEC, etc.

For preparation of polyclonal antibodies, the first step is immunizationof the host animal with the target protein, where the target proteinwill preferably be in substantially pure form, comprising less thanabout 1% contaminant The immunogen may comprise the complete targetprotein, fragments or derivatives thereof. To increase the immuneresponse of the host animal, the target protein may be combined with anadjuvant, where suitable adjuvants include alum, dextran, sulfate, largepolymeric anions, oil& water emulsions, e.g. Freund's adjuvant, Freund'scomplete adjuvant, and the like. The target protein may also beconjugated to synthetic carrier proteins or synthetic antigens. Avariety of hosts may be immunized to produce the polyclonal antibodies.Such hosts include rabbits, guinea pigs, rodents, e.g. mice, rats,sheep, goats, and the like. The target protein is administered to thehost, usually intradermally, with an initial dosage followed by one ormore, usually at least two, additional booster dosages. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst the mouse protein, the animal will generally be a hamster,guinea pig, rabbit, etc. The antibody may be purified from the hybridomacell supernatants or ascites fluid by conventional techniques, e.g.affinity chromatography using GST-3 bound to an insoluble support,protein A sepharose, etc.

The antibody may be produced as a single chain, instead of the normalmultimeric structure. Single chain antibodies are described in lost etal. (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding thevariable region of the heavy chain and the variable region of the lightchain are ligated to a spacer encoding at least about 4 amino acids ofsmall neutral amino acids, including glycine and/or serine. The proteinencoded by this fusion allows assembly of a functional variable regionthat retains the specificity and affinity of the original antibody.

For in vivo use, particularly for injection into humans, it is desirableto decrease the antigenicity of the antibody. An immune response of arecipient against the blocking agent will potentially decrease theperiod of time that the therapy is effective. Methods of humanizingantibodies are known in the art. The humanized antibody may be theproduct of an animal having transgenic human immunoglobulin constantregion genes (see for example International Patent Applications WO90/10077 and WO 90/04036). Alternatively, the antibody of interest maybe engineered by recombinant DNA techniques to substitute the CH1, CH2,CH3, hinge domains, and/or the framework domain with the correspondinghuman sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes isknown in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J.Immunol, 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtrscription termination occur at native chromosomal sites downsteam ofthe coding regions. The resulting chimeric antibody may be joined to anystrong promoter, including retroviral LTRs, e.g. SV40 early promoter,(Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcoma virus LTR(Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murine leukemiavirus LTR (Grosschedl et al (1985) Cell 41:885); native Ig promoters,etc.

In yet other embodiments of the invention, the active agent is an agentthat modulates, and generally decreases or down regulates, theexpression of the gene encoding the target protein in the host. Forexample, antisense molecules can be used to down-regulate expression ofGST-3 in cells. The anti-sense reagent may be antisense oligonucleotides(ODN), particularly synthetic ODN having chemical modifications fromnative nucleic acids, or nucleic acid constructs that express suchanti-sense molecules as RNA. The antisense sequence is complementary tothe mRNA of the targeted gene, and inhibits expression of the targetedgene products. Antisense molecules inhibit gene expression throughvarious mechanisms, e.g. by reducing the amount of MRNA available fortranslation, through activation of RNAse H, or steric hindrance. One ora combination of antisense molecules may be administered, where acombination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840-844).

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al. (1993), supra, and Milligan et al.,supra.) Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5- propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g. ribozymes, anti-sense conjugates, etc. may be used toinhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. (1995), Nucl Acids Res. 23:4434-42). Examples of oligonucleotideswith catalytic activity are described in WO 9506764. Conjugates ofanti-sense ODN with a metal complex, e.g. terpyridyl Cu(II), capable ofmediating mRNA hydrolysis are described in Bashldn et al. (1995), Appl.Biochem. Biotechnol. 54:43-56.

As mentioned above, an effective amount of the active agent isadministered to the host, where “effective amount” means a dosagesufficient to produce a desired result. Generally, the desired result isat least a reduction in the amount of selectin binding as compared to acontrol.

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredinhibition of selectin binding. Thus, the agent can be incorporated intoa variety of formulations for therapeutic administration. Moreparticularly, the agents of the present invention can be formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols.

As such, administration of the agents can be achieved in various ways,including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such as crytlinecellulose, cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators, such as corn starch, potato starch or sodiumcarboxymethylcellulose; with lubricants, such as talc or magnesiumstearate; and if desired, with diluents, buffering agents, moisteningagents, preservatives and flavoring agents.

The agents can be formulated into preparation for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilzers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the lie.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonfull, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Where the agent is a polypeptide, polynucleotide, analog or mimeticthereof, e.g. antisense composition, it may be introduced into tissuesor host cells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al. (1992),Anal Biochem 205:365-368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152-154), where gold microprojectiles arecoated with the therapeutic DNA, then bombarded into skin cells.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven compound are readily determinable by those of skill in the art bya variety of means.

The subject methods find use in the treatment of a variety of differentdisease conditions involving selectin binding interactions, particularlyL-, E- or P- selectin, and more particularly L-selectin mediated bindingevents. Such disease conditions include those disease conditionsassociated with or resulting from the homing of leukocytes to sites ofinflammation, the normal homing of lymphocytes to secondary lymphorgans; and the like. Accordingly, specific disease conditions that maybe treated with the subject methods include: acute or chronicinflammation; autoimmune and related disorders, e.g. systemic lupuserythematosus, rheumatoid arthritis, polyarteritis nodosa, polymyositisand dermatomyositis, progressive systemic sclerosis (diffusescleroderma), glomerulonephritis, myasthenia gravis, Sjogren's syndrome,Hashimoto's disease and Graves' disease, adrenalitis,hypoparathyroidism, and associated diseases; pernicious anemia;diabetes; multiple sclerosis and related demyelinating diseases; uveitispemphigus and pemphigoid; cirrhosis and other diseases of the liver;ulcerative colitis; myocarditis; regional enteritis; adult respiratorydistress syndrome; local manifestations of drug reactions (dermatitis,etc.); inflammation-associated or allergic reaction patterns of theskin; atopic dermatitis and infantile eczema; contact dermatitis,psoriasis lichen planus; allergic enteropathies; atopic diseases, e.g.allergic rhinitis and bronchial asthma; transplant rejection (hearkidney, lung, liver, pancreatic islet cell, others); hypersensitivity ordestructive responses to infectious agents; poststreptococcal diseasese.g. cardiac manifestations of rheumatic fever, etc.; tissue rejectionduring tmnsplantation; and the like.

By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as inflammation and painassociated therewith. As such, treatment also includes situations wherethe pathological condition, or at least symptoms associated therewith,are completely inhibited, e.g. prevented from happening, or stopped,e.g. terminated, such that the host no longer suffers from thepathological condition, or at least the symptoms that characterize thepathological condition.

A variety of hosts are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the hosts will be humans.

Kits with unit doses of the active agent, usually in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

The following examples are offered primarily for purposes ofillustration It will be readily apparent to those skilled in the artthat the formulations, dosages, methods of administration, and otherparameters of this invention may be further modified or substituted invarious ways without departing from the spirit and scope of theinvention.

EXPERIMENTAL

I. Identification of GST-1 & GST-2

Human ESTs that are related to the C6ST/KSST at the protein level weresearched by using TBLASTN which compares a protein query sequenceagainst a nucleotide sequence database translated in all 6 readingframes. See Karlin, Samuel and Stephen F. Altschul (1990), Methods forassessing the statistical significance of molecular sequence features byusing general scoring schemes, Proc. Natl. Acad. Sci. USA 87:2264-68;and Karlin, Samuel and Stephen F. Altschul (1993), Applications andstatistics for multiple high-scoring segments in molecular sequences,Proc. Natl. Acad. Sci. USA 90:5873-7. As shown in Table 1, several ESTs,ranging from 228 to 861 bases, resulted in high scores. When comparedover their entire length with the C6ST/KSST, the predicted amino acididentities ranged from 27% to 57%.

TABLE 1 Human ESTs related to the chick chondroitin 6/keratan sulfatesulfotransferase Contig Covering AA of Identity of AA Assign- No. mRNAsource C6ST/KSST Sequences (%) ment 1 infant brain 347-451 42 1 2 infantbrain 140-216 57 1 3 adult heart 405-451 42 1 4 fetal lung  89-375 30 25 fetal liver/spleen 332-403 27 2 6 teratocarcinoma 100-165 31 2The cDNA clones corresponding to each EST were obtained from the ATCCand Research Genetics, Inc.,(Huntsville, Ala.) and sequenced in fillu toobtain further 3′ information. Sequence alignment analysis revealed thepresence of two distinct sequences (“contigs”), covering 74% (contig 1,starting at amino acid 137) and 78% (contig 2, starting at amino acid100). Contig sequences 1 and 2 are apparently both complete at the 3′end, since both contain poly A tracts at the end of their 3′untranslated regions (UTR).

Expression of transcripts corresponding to the two contigs was examinedin a number of human tissues by Northern analysis. Blots of poly A⁺ RNA(Clontech, Palo Alto, Calif.) were probed at high stringency with probesderived from the EST clones. A 3.1 kb band corresponding to contig I wasdetected in multiple human organs (heart, placenta, lung, liver,skeletal muscle, kidney, pancreas, spleen, lymph nodes, thymus) but moststrongly in brain. Contig 2 was also broadly expressed in various organs(3.3 kb band), including lymph node and brain.

Full-length cDNAs containing the two contigs and predicting CS6T/KSSThomologs were obtained by screening a human fetal brain λZAP cDNAlibrary (Stratagene, La Jolla, Calif.) with labeled 700-800 bprestriction fragments (from EST 2 for contig 1 and from EST 5 for contig2). Briefly, 10⁶ plaque-forming units were used to infect E. coli, whichwere then distributed on 20 plates. Duplicate filter lifts wereperformed. The probes were labeled with ³²P by random priming(Amersham), and hybridization was performed at 60° C. with highstringency washing. In both screens, multiple positive spots wereobtained in the first round. Single positive clones were obtained aftereither the second or third round of screening. Multiple clones weresequenced for each gene and the presence of the ESTs was confirmed Aswill be described below, the cDNAs contain open reading frames thatencode predicted proteins of high homology to CS6T/KSST. The proteinsencoded by these cDNAs were designated as GST 1 and GST 2, where “GSI”denotes “glycosylsulfotransferase.” GST 1 has been independently clonedand assigned the name “KSGal6ST) by Fukuta et al., J. Biol. Chem. (1997)272: 32321-8.

II. Identification of GST-3

ESTs potentially coding for novel human glycosyl sulfotransferases otherthan GST-1 & 2 were identified through a secondary homology screen, inwhich the peptide sequences of GST-1 and GST-2 were used as template intwo parallel TBLASTN searches against a public (dbest) and a privategenomic database (Lifeseq, Incyte Pharmaceuticals, Palo Alto, Calif.).Only matches that produced alignments with smallest sum probabilitiesP(N)<10⁻⁵ were selected from the output of the search, imported into acontig assembler (Sequencher 3.0, Gene Codes Corporation, Ann Arbor,Michigan) and assembled using the default settings of the program Thevast majority of these matches assembled into two contigs defined byGST-1 and GST-2. However, four particular ESTs found only in the privateLifeseq database did not assemble into either contig or with each other.These were termed GST-3 through GST6.

III. GST-3 is Expressed in High Endothelial Venules

In order to investigate if any of the above putative human glycosylsulfotransferases or similar genes were expressed in high endothelialvenules, an HEV-derived cDNA pool for use as template in homologypolymerase chain reaction (PCR) was prepared. In order to clone HEVgenes, an expression library from the aforementioned HEV-derived cDNAwas also generated. Briefly, total RNA (45 μg) was isolated from 10⁷HEC. Since the amount of poly A⁺ PNA was too limited for preparation ofa cDNA library by conventional procedures, the Capfinder (SMAR™) cDNAtechnology (CLONTECH) was used. In this technique, the reversetranscription reaction is primed by a modified oligo(dt) primer(containing a Not I site) and a “SMART” oligonucleotide which anneals toan oligo dC stretch added by reverse transcriptase (RT) at the 3′ end ofthe first strand cDNA. The annealed oligonucleotide serves as a “switch”template for RT, resulting in the generation of single stranded cDNAswhich are enriched for full length sequences and contain universalprimer sites for subsequent long distance PCR amplification. Thistechnology therefore makes it possible to generate high quality doublestranded cDNA (from limiting amounts of RNA), which is sufficient toconstruct a library. According to the published test results for thistechnology, Capfinder cDNA is comparable to conventionally prepared cDNAin gene representation and is significantly enriched for full lengthcDNAs. The HEC cDNA generated by the Capfinder technology was evaluatedby PCR for the presence of the following genes, which are known orsuspected to be expressed in HEC: CD34 (Baumhueter et al., Science(1993) 262: 436438), hevin (Girard & Springer, lmmunity (1995)2:113-123), fucosyltransferase VII (Maly et al., Cell (1996) 86:643653); β-1,6-N-acetylglucosarinyltransferase (C2GnT) (Bierhuizen &Fukuda, Proc. Natl. Acad. Sci. USA (1992) 89: 9326-9330), andfractalkine (Schall, Immunology Today (1997) 18:147). By this analysis,all of these cDNAs were detected in the HEC cDNA, and at least two ofthem (CD34 and C2GnT) were full length. With this validation of the HECcDNA, a library was generated as follows: the double-stranded cDNA wasligated to Eco RI adapters, digested with Not I and cloned into the NotI and Eco RI sites of pCDNA1.1 (Invitrogen, Inc, Carlsbad, Calif.),which is a modified version of the eucaryotic expression vector pCDM8(Aruffo et al., Proc. Natl. Acad. Sci. USA (1987) 84: 8753-8577). Theresulting libary has a complexity of 500,000 independent clones and anaverage insert size of 1.1 kb, according to the characterizationperformed by CLONTECH.

HEV-derived Capfinder cDNA was used as a template for homology PCR withdegenerate primers. In-frame translations of GST-1 and GST-2 werealigned with other known sulfotransferase protein sequences retrievedfrom the public databases. See FIG. 3. Three putative consensus regionswere identified, and the following degenerate primers were synthesizedto encode within these consensus regions a maximal number of possiblepermutations at the amino-acid level in order to cover a maximal numberof novel sulfotransferases that may fall into these patterns.

These primers were (I=inosine):

A+: 5′ TWYTWYCTITWYGARCCICTITGGCAYST 3′ (SEQ ID NO:03) B+: 5′CTIAAICTISTICWRCTISTIMGIRAYCC 3′ (SEQ ID NO:04) B−: 5′GGRTYICKIASIAGYWGIASIAGITTIAG 3′ (SEQ ID NO:05) C−: 5′AGRTCYTCRTAICKIAGIAGIAKRTA 3′ (SEQ ID NO:06)

In the first round PCR each reaction contained in a total volume of 50μl 100 mM Tris-Cl (pH 8.3), 0.5 mM KCl, 15 mM MgCl₂, forward and reverseprimer (0.5 μM each), dATP, dCTP, dGTP, and dTTP (100 μM each), 0.25units Thermus aquaticus DNA polymerase (Boehringer Mannheim #1647679),and 0.5 μl of HEV-message derived Cap-finder cDNA preparation (generatedby Clontech Inc., cf. above). In “no template” control samples the cDNAwas omitted.

Each reaction was cycled as follows: hold 4 min @ 94° C., then 35 cyclesof [30 sec @ 94° C. followed by 30 sec @ 40° C. followed by 1 min @ 72°C.], then hold 6 min @ 72° C. Following completion of PCR a 20 μlaliqout of each reaction was analysed by standard horizontal agarose(1%) gel electrophoresis. No discernable band pattern was observed (datanot shown).

Therefore the unfractionated products of the first round PCR were usedas template in a second round PCR. Here each reaction contained in atotal volume of 50 μl 100 mM Tris-Cl (pH 8.3), 0.5 M kCl, 15 mM MgCl₂,forward and reverse primer (0.5 μM each), dATP, dCITP, dGTP, and dTTP(100 μM each), 0.25 units Thermus aquaticus DNA polymerase (BoehringerMannheim #1647679), and 1 μl of total PCR reaction from round 1 (cf.above).

Each reaction was cycled as follows: hold 4 min @ 94° C., then 35 cyclesof [30 sec @ 94° C. followed by 30 sec @ 45° C. followed by 1 min @ 72°C.], then hold 6 min @ 72° C. The entire reactions were thenfractionated by standard horizontal agarose (1%) gel electrophoresis.Three bands appearing at positions 2.1, 2.2 and 2.3, were excised andDNA eluted from the gel using the QIAquick PCR purification kit (QiagenInc. #28104). Eluted DNA was then subcloned into the TA cloning vectorpCR-II (stratagene) and E-coli transformed with recombinant plasmids.For each band eight colonies were expanded, and plasmid DNAs isolatedand sequenced using standard dideoxynucleotide chain terminationmethodology with fluorimetric detection.

In order to map the amplicons generated by the above homology PCR,public (dbest) and private (Incyte Inc.) EST databases were screenedwith by the TBLASTX algorithm (Karlin & Altschul, 1990 & 1993; cf.above) using the sequences of these amplicons as query sequences. Foursequences amplified from from HEV-cDNA with primers B+ and C− alignedwith >95% overall identity to Incyte EST #2620445 defined perviously asGST-3 (cf. above). All other query sequences did not pick upstatistically significant matches in the specified databases.

IV. GST-3 is Expressed in HEC

From the extended DNA sequence of Lifeseq clone #2620445=GST-3 wedesigned a nondegenerate primer pair located within the incomplete openreading frame encoded by this EST.

Forward: 5′ AAACTCAAGAAGGAGGACCAACCCTACTATGTGATGC 3′ (SEQ ID NO: 07)Reverse: 5′ ATAAAGCTTGTGGATTTGTTCAGGGACATTCCAGGTAGACAGAAGAT 3′ (SEQ IDNO: 08)

Using RT-PCR, a PCR product of appropriate length (500 bp) was amplifiedfrom HEC cDNA with this primer pair. This product could not be amplifiedfrom cDNAs prepared from tonsillar lymphocytes or primary cultured humanumbilical vein endothelial cells (HUVEC). Control primers forhypoxanthine phosphoribosyl tnansferase (HPRT, a ubiquitously expressedcellular “housekeeping enzyme”) were used in parallel to establish thatsimilar amounts of template were used in each set of PCR reactions andthat none of the template DNAs were substantially degraded. These RT-PCRresults confirm that the gene corresponding to the PCR product isexpressed in HEC but not in lymphocytes or HUVEC. Northern analysis hasfailed to detect mRNA for the new gene in a variety of human tissues andorgans (but did detect a signal in liver, pancrease, lymph node and HEC,establishing that the expression of this gene is highly restricted.Also, by in situ hybridization, transcripts for mouse GST-3 wereselectively found in lymph node HEV.

V. GST-3 Cloning

A full length cDNA from the HEC library described in the previoussection was cloned as follows. The pool selection procedure described inBakker et al., J. Biol. Chem. (1997) 272:29942-6) was used to quicklyisolate the cDNA. It was first established that the relevant templatewas contained within the library by successfully amplifying the abovedescribed PCR product from the library stock comprising the entirelibrary. An aliquot of this bacterial stock was then divided into 200pools of 2000-3000 colonies each. Each pool was plated out on LB platesand the colonies were allowed to grow to a healthy size. The colonieswere harvested in LB and allowed to grow further at 37° C., at whichtime glycerol stocks were prepared from each pool. By PCR analysis ofthe pools, nine positives were identified in this first round ofscreening. The corresponding bacterial stock for one of these pools wasthen titered and plated at 100 colony forming units (cfu) per plate in40 plates. Plates were grown, harvested, preserved and analyzed as inthe first round, resulting in the identification of three positivesubpools. At this stage, one of the three positive pools was plated at adensity (300 cfu) so that individual colonies could be analyzed by PCROne cDNA clone was obtained by this approach. It contains a completeopen reading frame which encodes a novel 386 amino acid protein, termedGST-3. This fill length cDNA sequence was then used as template in aBLASTN search of the public (dbest) and Lifeseq EST databases. In thismanner, two so far unrecognized ESTs #2617407 (from Lifeseq; derivedfrom a human gall bladder cDNA library) and g2262929 (from the mouse ESTcollection included in the dbest database, derived from a murine mammarygland cDNA library) were identified. The former EST included the 5′ endof GST-3 open reading frame. Since this EST was generated with an oligodT-primer, it therefore contains the entire open reading frame plus all3′ untranslated sequence of the human GST-3 cDNA. This EST was retrievedfrom Incyte in the form of a plasmid-transformed E. coli culture,expanded into Luria Bertoni Medium (with 0.1 mg/ml Ampicillin). Theplasmid was isolated from a 500 ml culture and sequenced using standarddideoxynucleotide chain termination methodology with fluorimetricdetection. Since no PCR-step was used in generating the full lengthGST-3 Lifeseq EST Incyte #2167407 (in contrast to the Capfindermethodology employed in generation of our HEV-library), the GST-3sequence obtained from Incyte #2617407 is free of PCR errors. Thesequence is provided in SEQ ID NO:02 and shown in FIG. 1.

VI. Characterization of GST-3

A. Three cDNA clones which encode three different human homologs forC6ST/KSST have been obtained The predicted GST proteins are type 2membrane proteins 411, 484, and 386 amino acids in length, respectively.Each has a relatively short transmembrane domain and a shortamino-terminal cytoplasmic tail. Table 2 demontrates the high homologiesamong the 3 human proteins and the chick CS6T/KSST. Overall homologiesat the amino acid level ranged from 28% to 40% identity. Strikingly,there are three regions of 16 to 29 amino acids in which identity amongthe three GSTs ranged from 50-59% and similarity ranged from 65-94%. SeeFIG. 4. FIG. 4 shows that all four of the sulfotransferases are type IItransmembrane proteins with short cytoplasmic tails (TM). There arethree regions (region A, B and C) in which identities among the humanGSTs range from 50-59% and similarities range from 65 to 94%. The aminoacid sequence for the regions are:

-   A:    (T/S)XRSGSSF(V/F)G(Q/E)LFXQX(P/L)(D/E)VF(F/Y)L(F/Y/M)EP(L/V/A)(W/Y)HV-   B: L(N/D)L(K/H)(V/I)(I/V)XLVRDPR(A/G)(V/I)(LAF)-   C: PXXL(Q/K)XXY(L/M)(L/V)VRYEDL(A/V)XXP

TABLE 2 Percent amino acid identities for the predicted coding sequencesGST 1 GST 2 GST 3 CS6T/KSST GST 1 — 31 32 40 GST 2 — — 35 28 GST 3 — — —31

B. GST-1 is the same as the sulfotransferase reported by Fukuta et al.supra (1997) and named KS Gal6ST. GST-3 (HEC-GlcNAc6 ST), is a novelGlcNAc-6-sulfotransferase. These two sulfotransferase, together with thechicken C6/KSST (Fukuta et al., 1995, supra) and the recently reportedhuman chondroitin-6-sulfotransferase (C6ST, specificity for C-6 ofGalNAc, Fukcuta, et al., Biophys. Act. (1998) 1399:57-6 1) andGlcNAc-6-sulfotransferase (GlcNAc6ST) (Uchimura et al., J. Biochem(Tokyo)(998b)124670-678) (which corresponds to GST-2) constitute afamily of highly conserved enzymes. Overall amino acid identities withthe family range from 27 to 42%. These enzymes are type II transmembraneproteins with short cytoplasmic tails, features which are typical ofglycosyltransferases and carbohydrate sulfotransferases, with theexception of the heparan sulfate D-glucosamino-3-O-sulfotransferase(Shworak et al., 1997).

Within this new family of carbohydrate sulfotransferases, there arethree regions of *amino acid sequence in which amino acid identityranges from 45-54% and similarity from 80-90% (FIG. 5). Regions one andtwo contain elements that conforming to the recently described consensusbinding motifs for the high energy phosphate donor,3′-phosphoadenosine-5′-phosphosulfate. These elements are found in allsulfotransferases characterized to date (Kakuta et al., Trends Biochem.Sci. (1998)23:129-130. In addition, regions one and three contain twostretches of sequence of 11 amino acids each (corresponding to aminoacids 124-134 and 328-339, respectively, in the HEC-GlcNAc6ST sequence)that are highly conserved (>90% similarity). This suggests that thesetwo elements contribute to a binding pocket that interacts with the6-hydroxyl group of an appropriate oligosaccharide acceptor (Gal,GalNAc, or GlcNAc) to bring it into apposition with the donorphosphosulfate group.

VII. Characterization of GST-3 (HEC-GlcNAc6STt) and KSGal6ST

A. GST-3 Sulfates GlyCAM-1

In expression experiments, the sulfotransferase activity of the GST 3protein by transient expression of its cDNA into COS cells has beeninvestigated. Since the HEC library yielding the GST 3 cDNA was in thepcDNA1.1 expression vector, there was no need to subclone the GST 3insert prior to transfection. Co-transfection of the GST 3 cDNA with acDNA encoding a GlyCAM-l1human IgG1 Fc chimera resulted in a >10 foldenhanced incorporation of ³⁵S—SO₄ relative to transfection with theGlyCAM-1 chimera alone. Co-transfection with vector cDNA had no effect.By SDS-PAGE analysis, incorporation of ³⁵S—SO₄ counts into the GlyCAM-1chimera was confirmed. The results are shown in FIG. 6. The resultsindicate that GST 3 encodes a sulfotransferase that can utilize GlyCAM-1as an acceptor.

B. Specificity of GST-3 (HEC-GlcNAc6ST) Defined with Synthetic Acceptors

To define the activity of the protein encoded by the novel cDNAdescribed above, the novel cDNA was expressed in COS cells and cellularextracts were tested for their ability to transfer ³⁵S-sulfate from35S-[3′ phosphoadenosine 5′ phosphosulfate] (PAPS) to syntheticoligosaccharide acceptors using the assay described in Bowman et al.,Chem. and Biology (1998) 5:447-460. The disaccharide and trisaccharideacceptors (Table 3) were based on core structures of GlyCAM-1 chains(FIG. 7) with the substitution of Gal for GalNAc at the reducingtermini.

TABLE 3 Nomenclature and Structure of Oligosaccharides and SubstituentsName Structure sLe^(x) (sialyl Lewis x) Siaα2→3Galβ1→4[Fucα1→3]GlcNAc6′-sulfo sLe^(x) Siaα2→3[SO₃→6]Galβ1→4[Fucα1→3]GlcNAc 6-sulfo sLe^(x)Siaα2→3Galβ1→4[Fucα1→3][SO₃→6]GlcNAc 6′,6-disulfo sLe^(x)Siaα2→3[SO₃→6]Galβ1→4[Fucα1→3] [SO₃→6]GlcNAc core 2Galβ1→3[GlcNAcβ1→6]GalNAc disaccharide GlcNAcβ1→6Galα1-R acceptortrisaccharide Galβ1→4GlcNAcβ1→6Galα1-R acceptor RC—CH₂—CH₂CONH₂—(CH₂)₇—CH₃

Substantial radioactivity was transferred to the disaccharide acceptorGlcNAcβ1-6Galα-R, but the activity towards the trisaccharide acceptorGalβ1-4GlcNAcβ1-6Galα-R was barely above the control generated by anextract from mock-transfected cells (FIG. 8). The position andregiochemistry of sulfation was established by HPAEC analysis using apreviously described protocol (Bowman et al., 1998, supra). Label wasassociated exclusively with GlcNAc-6-sulfate (FIG. 9). This analysisestablishes that this cDNA encodes a GlcNAc-6-sulfotransferase,providing the basis for the designation HEC-GlcNAc6ST. Furthermore, theenzyme requires a terminal GlcNAc residue for recognition, mirroring theprofile of activity observed previously in extracts of isolated HEC(Bowman et al., 1998, supra).

C. Sulfation of GlyCAM-1 by KSGal6ST (GST-1) and HEC-GlcNAc6ST (GST-3)

To test whether KSGal6ST and HEC-GlcNAc6ST were capable of sulfating abonafide L-selectin ligand in cells, COS cells were transfected with acDNA encoding a GlyCAM-1/IgG chimera and a cDNA encoding one or theother sulfotransferase. The transfected cells were cultured in thepresence of ³⁵S-sulfate and radiolabeled GlyCAM-1/Ig was purified fromthe conditioned medium on protein A-agarose. It was found thatsubstantial incorporation of counts occurred when GlyCAM-1/IgG (66 kDa)was cotransfected with either KSGal6ST or HEC-GlcNAc6ST cDNA but notwith the empty vector.

In order to establish the regiochemistry of sulfation on radiolabeledGlyCAM-1/IgG, samples resulting from the two sulfotransferasetransfections were subjected to hydrolysis and analysis by HPAEC, usinga Dionex HPLC system according to the previously established procedures(Hemmerich et al., Biochem. (1994)33:4820-4829). As shown in FIG. 10,the retention times of the released sulfated mono- and disaccharidescorresponded to those of authentic standards for [SO₃→6]Gal and[SO₃→6]Galβ1→4GlcNAc for the KSGal6ST transfectants and to [SO₃→6]GlcNAcand Galβ1→4[SO₃→6]GlcNAc for the HEC-GlcNAc6ST transfectants. Thus, thespecificities of KSGal6ST and HEC-GlcNAc6ST for oligosaccharides ofGlyCAM-1 reflected those observed with the model acceptors (FIGS. 8 & 9,Fukuta et al., 1997).

D. Contribution of KSGal6ST and HEC-GlcNAc6ST to the Generation ofL-selectin Ligand Activity

In order to test whether KSGal6ST and HEC-GlcNAc6ST can contribute tothe generation of L-selectin ligand activity, the binding of anL-selectin/IgM chimera to CHO cells which were transiently transfectedwith cDNAs for the sulfotransferases and a cDNA encoding CD34 wasexamined The recipient CHO cells, termed CHO/FTVII/C2GnT, were stablytransfected with 1) flicosyltransferase VII (FucTVII), which is known tobe involved in the biosynthesis of L-selectin ligands (Maly et al., Cell(1996) 643-653); and 2) core 2 β1→6 N-acetylglucosaminyltransferase(C2GnT) (Bierhuizen and Fukuda, Proc. Nat'l Acad. Sci. USA(1992)89:9326-9330). This latter enzyme provides a core structure forO-linked glycans upon which extended chains with sLe^(x) capping groupsare elaborated (Li et al., J. Biol. Chem (1996) 271:3255-3264. Bindingwas measured by flow cytometry. As shown in FIG. 11 a, no L-selectin/IgMstaining above background was observed in the CHO cell transfectants inthe absence of sulfotransferase cDNA. Strong L-selectin/IgM binding wasobserved in a significant population of the cells when bothHEC-GlcNAc6ST and KSGal6ST cDNAs were included in the transfection. Thebinding of the L-selectin/IgM chimera was specific as indicated by itscalcium dependence (data not shown) and the inhibition of its binding bya function-blocking anti-L-selectin mAb (MEL-14) (FIG. 11 a). Thebinding of L-selectin/IgM was dependent on the presence of the CD34protein scaffold, as indicated by the nearly complete loss of stainingwhen the CD34 cDNA was omitted from the transfection (FIG. 11 b). Todetermine the individual contribution of the two sulfotransferases toL-selectin binding, we cotransfected the CHO cells were cotransfectedwith cDNAs for 1) CD34 and 2) the sulfotransferases, alone or incombination. Transfection with either HEC-GlcNAc6ST or KSGal6ST cDNAconferred binding of the L-selectin/IgM chimera (FIG. 11 c). KSGal6STappeared to exert the greater effect, both in terms of the proportion ofpositive cells and their mean fluorescence intensity (FIG. 11 c).However, the combination of KSGal6ST and HEC-GlcNAc6ST cDNAs stronglyenhanced the binding of L-selectin/IgM relative to the the singlesulfotransferase transfectants (FIG. 11 c). Moreover, the signalresulting from the combination clearly exceeded the sum of the signalsfrom the individual transfections, indicating that the twosulfotransferases synergized to generate ligand activity. Thissynergistic effect was evident over a range of cDNA concentrations inthe transfection mixtures (Table 4).

TABLE 4 L-Selectin/IgM Staining of CHO Cells Transfected withCombinations of Sulfotransferase cDNAs μg cDNA transfected MFI KSGal6STHEC-GlcNAc6ST L-sel-Igm Staining 1.0 0 364 1.5 0 391 0 1 114 0 1.5 1630.5 0.5 935 1 0.5 917 0.5 1.0 830 CHO/FTVII/C2GnT cells werecotransfected with plasmids encoding CD34 (2 μg) and eachsulfotransferase alone or in combination in the indicated amounts. Dataare expressed as the mean fluorescence intensity (MFI) of L-selectin/IgMstaining in the L-sel-IgM*/CD34* population with background signal (fromtransfectants with CD34 cDNA alone, value 139) subtracted.VIII. Rolling Data in a Parallel Plate Flow Chamber

A. Methods

1. Transient Transfection of COS-7 Cells

For generation of recombinant GlyCAM-1/IG fusion protein, COS-7 cellswere grown to 80% confluency in 10 cm culture dishes (Nunc) andtransfected with plasmids encoding core 2β-1,6-N-acetylglucosaminyltransferase (C2Gnt) (1 μg), fucosyltransferaseVII (FT-VII) (1 μg), GlyCAM-1/IgG (2 μg) and 0.5, 1 or 2 μg of eitherKSGal6ST, huGlcNAc6ST, HEC-GlcNAc6ST, or irrelevant control plasmid(mock control), using Lipofectamine (Life Technologies) in Opti-MEM(Life Technologies) according to the manufacturer's protocol. Cells weregrown for seven days in Opti-MEM/penicillin/streptavidin. RecombinantGlyCAM-1/IgG fusion protein was isolated from the conditioned medium(CM) by affinity chromatography on protein A-agarose and transferred toPBS on a Centricon 30 concentrator (Amicon, Beverly, Mass.).

2. Laminar Flow Assays

The GlyCAM-1/IgG constructs were coated at similar site densities ontoolystyrene dishes (Corning, San Mateo, Calif.) as determined by ELISAusing 96 well polystyrene plates (Costar, Corning, N.Y.). Proteins werecoated in Tris-buffered saline (TBS), pH 9.0 overnight at 4° C., washedand blocked with 3% BSA. The immobilized GlyCAM-1/IG constructs weredetected by ELISA with biotinylated CAMO-5 or biotinylated anti humanIgG (Fc specific) and streptavidin conjugated alkaline phosphatase. Forflow experiments the substrate-coated dishes were incorporated as thelower wall of a parallel plate flow chamber (Lawrence et al., Cell(1991) 65:859-873) and mounted on the stage of an invertedphase-contrast microscope microscope (Diaphot TMD; Nikon Inc., GardenCity, N.Y.). Jurkat cells were perfused through the flow chamber at1-2×10⁶ cells/ml in HBSS with Ca⁺ or Mg⁺ supplemented with 0.2% BSA. Forinhibition studies, cells were treated with 5 μg/ml DREG56 (antiL-selectin mAb), 10 μg/ml Fucoidin (a carbohydrate inhibitor ofL-selectin) for 10 min at room temperature prior to injection into theflow chamber or perfused in 5mM EDTA/HBSS/0.2% BSA. In some cases,substrates were incubated with 5 mU/ml Vibrio cholera sialidase (OxfordGlycosystems, Rosedale, N.Y.) for 30 min in 50 mM sodium acetate, 4 mMCaCl₂, 0.1% BSA, pH 5.5 or as a control with buffer alone.

Experiments were videotaped using a 4x objective (field of view −1.91mm²) and data were analyzed n a computer using NIH Image 1.61. Fortethering, cells were perfused through the chamber at different shearstresses covering a range from 3 dynes/cm² to 0.2 dynes/cm². Thefraction of cells that came into close proximity with the substrate andtethered stably (cells that continued to roll for >1s on the substrateafter the initial attachment) was determined. For rolling velocities,cells were infused for 2 min at 1 dyne/cm² after which shear stress wasincreased in 1.5 to twofold increments up to 35 dynes/cm² in intervalsof 5 s (detachment assay). Cell displacement was followed for 1-3seconds to determine rolling velocities at each shear stress. Indetachment assays the number of rolling cells at each shear stress wasdetermined. The maximum number of adherent cells was set to 100%, andthe number of rolling cells at higher shear stresses was expressedrelative to this peak accumulation value. In all experiments theobservation field was located at the upstream edge of the spot ofadsorbed protein to minimize contribution of cells rolling into thefields from upstream fields.

B. Results

1. Jurkat Cells Roll on Fucosylated and Sulfated GlyCAM-1/IgG in ShearFlow

The binding of L-selectin to its HEV-ligands requires sialylation,fucosylation (Maly et al., 1996) and sulfation. Maly et al. establishedthat FucT-VII is the relevant fucosyltransferase for the elaboration offunctional L-selectin ligands in HEC. Our previous structural analysisindicated that Gal-6sulfate and GlcNAc-6sulfate are equally present innative GlyCAM-1 oligosaccharides and that both reside within thesLe^(x)-based capping structures (6′-sulfo sLe^(x) and 6-sulfo ssLe^(x), respectively). In order to test how these sulfationmodifications contribute to the generation of L-selectin ligand activitythe rolling of Jurkat cells on immobilized, flicosylated, and sulfatedrGlyCAM-1/IgG fusion proteins under shear flow conditions was examined.COS cells, which have endogenous sialyl transferase activity, werecotransfected with cDNAs for C2GnT, FucT-VII, GlyCAM-1/IgG and eitherKSGal6ST, huGlcNAc6ST, HEC-GLCNAC6ST or control plasmid RecombinantGlyCAM-1/IgG fusion proteins were purified from the conditioned mediumon protein A-agarose.

For flow assays the recombinant proteins were coated at equal sitedensities as determined by ELISA (data not shown). Detection of theimmobilized GlyCAM-1/IgG substrates with the anti-GlyCAM-1 antibodyCAMO-5 or an anti-human IgG (Fc specific) mAb demonstrated the samelevel of binding of the various chimeras. Rolling of Jurkat cells on thesubstrates at a shear stress of 1 dyne/cm² required the fucosylation ofGlyCAM-1/IgG, as no rolling interactions were observed on GlyCAM-1/IgGor sulfated, non-fucosylated GlyCAM-1/IgG (Table 5). Sulfation of C-6 ofeither Gal or GlcNAc yielded an increase in the number of rolling cells,however the effect was more pronounced for the Gal-6-sulfated substrate.An anti-L-selectin mAb abrogated the interaction with the substrate asdid fucoidin, an anionic polysaccharide that blocks L-selectin-dependentbinding. T tent of the substrates with sialidase from Vibrio choleracompletely prevented Jurkat cell tethering as previously seen forlymphocyte tethering onto PNAd and CD34 (Puri et al., 1995) or HEV(Rosen et al., 1985) (Table 5).

TABLE 5 Characteristics of the interaction of Jurkat cells withrGlyCAM-1/IgG fusion proteins anti L-selectin Fu- sial- substrateRolling¹ EDTA² mAb² coidin³ idase³ rGlyCAM-1/IgG 0 rGlyCAM-1/IgG 12.2 ±5   0 0 0 0 FT rGlyCAM-1/IgG 0 KSGal6ST rGlyCAM-1/IgG 0 huGlcNAc6STrGlyCAM-1/IgG 0 HEC-GlcNAc6ST rGlyCAM-1/IgG 107.0 ± 16.7 — — — — FTKSGal6ST rGlyCAM-1/IgG  36.1 ± 15.3 — — — — FT huGlcNAc6ST rGlyCAM-1/IgG36.6 ± 9.5 — — — — FT HEC-GlcNAc6ST ¹Jurkat cells were perfused throughthe flow chamber at 1 dyne/cm² at 1-2 × 10⁶ cells/ml and the number ofrolling cells was evaluated at 2 min of flow. ²Cells were treated with 5μg/ml DREG56, 10 μg/ml Fucoidin for 10 min at room temperature prior toinjection into the flow chamber or perfused in 5 mM EDTA/HBSS/0.2% BSAto inhibit L-selectin mediated rolling. ³Immobilized GlyCAM-1/Ig fusionproteins were treated with 5 mU/ml Vibrio cholera sialidase as describesin Methods. ⁴‘0’ indicates that no rolling of cells was detected.2. Sulfation of Fucosylated rGlyCAM-1 Enhances L-selectin MediatedInteractions in Shear Flow

To determine the contribution of sulfation to tethering under laminarflow conditions, Jurkat cells were perfused through a parallel wall flowchamber in which fucosylated GlyCAM-1/IgG constructs with or withoutsulfate modifications were coated at the same site densities. Jurkatcells showed the tethering profile characteristic for L-selectin with ashear threshold below which no or little tethering occurred (FIG. 12).While a maximum tethering rate of 9% was found for the interaction ofcells with non-sulfated GlyCAM-1/IgG (FT), the fraction of tetheredcells was doubled upon sulfation on C-6 of GlcNAc (FT, huGlcNAc6ST; FT,HEC-GlcNAc6ST) and increased sixfold upon sulfation C-6 of Gal (FT,KSGal6ST). Furthermore, the later modification resulted also in a shiftof the tethering threshold towards lower shear stresses (FIG. 12).

Sulfation of C-6 of GlcNAc reduced the velocity of rolling cellsconsistently relative to the velocities observed on non-sulfatedGlyCAM-I/IG. The reduction in velocity was more pronounced on the Gal-6sulfated substrate than on the substrates with GlcNAc modifications(FIG. 13). Gal-6 sulfation resulted in cells that were more resistant todetachment at increased shear stresses indicating an increase in bindingstrength (FIG. 14). Small increases in binding strength were observed onGlcNAc-6 modified substrates.

It is apparent from the above results and discussion that a novel humanglycosyl sulfotransferase, as well as polypeptides related thereto andnucleic acid compositions encoding the same are provided by the subjectinvention. These polypeptide and nucleic acid compositions find use in avariety of diverse applications, including research, diagnostic,screening and therapeutic applications. Also provided are improvedmethods of treating diseases associated with selectin-sulfated ligandmediated binding events, since agents that selectively reduce or inhibitthe activity of the subject enzyme are employed, so that othersulfotransferases whose activity is beneficial are not adverselyaffected.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A method for inhibiting a binding event between a selectin and aselectin ligand, said method comprising: contacting a cell that producessaid selectin ligand with an agent that inhibits the sulfation activityof a glycosyl sulfotransferase-3 (GST-3) polypeptide, wherein the GST-3polypeptide catalyzes the transfer of a sulfate group from a donorcompound to a selectin ligand precursor, wherein the GST-3 polypeptideis encoded by a nucleic acid comprising a sequence that is at least 75%identical to the sequence set forth in SEQ ID NO:2, and whereininhibition of said sulfation activity inhibits a binding event betweenthe selectin and the selectin ligand.
 2. The method of claim 1, whereinthe GST-3 polypeptide is encoded by a nucleic acid comprising a sequencethat is at least 90% identical to SEQ ID NO:2.
 3. The method of claim 1,wherein the GST-3 polypeptide is encoded by a nucleic acid comprising asequence that is at least 95% identical to SEQ ID NO:2.
 4. The method ofclaim 1, wherein the GST-3 polypeptide is encoded by a nucleic acidcomprising the nucleotide sequence as set forth in SEQ ID NO:2.
 5. Themethod of claim 1, wherein the selectin ligand is selected from thegroup consisting of an L-selectin ligand, a P-selectin ligand, and anE-selectin ligand.
 6. The method of claim 1, wherein the selectin is anL-selectin, and the selectin ligand is an L-selectin ligand.
 7. Themethod of claim 6, wherein said L-selectin ligand is selected fromGlyCAM-1, CD34, MAdCAM-1, Sgp200, and podocalyxin.
 8. The method ofclaim 1, wherein the cell is a high endothelial cell.
 9. The methodaccording to claim 1, wherein said agent is a small molecule.
 10. Themethod of claim 1, wherein said agent is an antibody specific for GST-3.11. The method of claim 10, wherein said antibody is a polyclonalantibody.
 12. The method of claim 10, wherein said antibody is amonoclonal antibody.
 13. A method for inhibiting a binding event betweena selectin and a selectin ligand, said method comprising: contacting acell that produces said selectin ligand with an agent that inhibits thesulfation activity of a glycosyl sulfotransferase-3 (GST-3) polypeptide,wherein the GST-3 polypeptide catalyzes the transfer of a sulfate groupfrom a donor compound to a selectin ligand precursor, wherein the GST-3polypeptide comprises an amino acid sequence that is at least 60%identical to the amino acid sequence set forth in SEQ ID NO:1, andwherein inhibition of said sulfation activity inhibits a binding eventbetween the selectin and the selectin ligand.
 14. The method of claim13, wherein the GST-3 polypeptide comprises the amino acid forth in SEQID NO:1.
 15. The method of claim 13, wherein the selectin ligand isselected from an L-selectin ligand, a P-selectin ligand, and anE-selectin ligand.
 16. The method of claim 13, wherein the selectin isan L-selectin, and the selectin ligand is an L-selectin ligand.
 17. Themethod of claim 16, wherein said L-selectin ligand is selected fromGlyCAM-1, CD34, MAdCAM-1, Sgp200, and podocalyxin.
 18. The method ofclaim 13, wherein the cell is a high endothelial cell.
 19. The method ofclaim 13, wherein said agent is a small molecule.
 20. The method ofclaim 13, wherein said agent is an antibody specific for GST-3.
 21. Themethod of claim 20, wherein said antibody is a polyclonal antibody. 22.The method of claim 20, wherein said antibody is a monoclonal antibody.23. A method for inhibiting a binding event between a selectin and aselectin ligand, said method comprising: contacting a glycosylsulfotransferase-3 (GST-3) polypeptide with an agent that inhibitssulfation activity of the GST-3 polypeptide, wherein the GST-3polypeptide catalyzes the transfer of a sulfate group from a donorcompound to a selectin ligand precursor, wherein the GST-3 polypeptidecomprises an amino acid sequence that is at least 60% identical to thesequence set forth in SEQ ID NO:1, and wherein inhibition of saidsulfation activity inhibits a binding event between the selectin and theselectin ligand.
 24. The method of claim 23, wherein the GST-3polypeptide comprises the amino acid sequence as set forth in SEQ IDNO:1.
 25. The method of claim 23, wherein said amino acid sequence isencoded by a nucleic acid comprising a nucleotide sequence that is atleast 75% identical to SEQ ID NO:2.
 26. The method of claim 23, whereinsaid amino acid sequence is encoded by a nucleic acid comprising anucleotide sequence that is at least 90% identical to SEQ ID NO:2. 27.The method of claim 23, wherein said amino acid sequence is encoded by anucleic acid comprising a nucleotide sequence that is at least 95%identical to SEQ ID NO:2.
 28. The method of claim 23, wherein the GST-3polypeptide is encoded by a nucleic acid comprising the nucleotidesequence as set forth in SEQ ID NO:2.
 29. The method of claim 23,wherein the selectin ligand is selected from the group consisting of anL-selectin ligand, a P-selectin ligand, and an E-selectin ligand. 30.The method of claim 23, wherein the selectin is an L-selectin, and theselectin ligand is an L-selectin ligand.
 31. The method of claim 30,wherein said L-selectin ligand is selected from GlyCAM-1, CD34,MAdCAM-1, Sgp200, and podocalyxin.
 32. The method according to claim 23,wherein said agent is a small molecule.
 33. The method of claim 23,wherein said agent is an antibody specific for GST-3.
 34. The method ofclaim 33, wherein said antibody is a polyclonal antibody.
 35. The methodof claim 33, wherein said antibody is a monoclonal antibody.